The incidence of life-threatening fungal infections is increasing at an alarming rate. With the exception of Staphylococci infections, the fungus C. albicans represents the fastest growing area of concern in hospital acquired infections. About 90% of nosocomial fungal infections are caused by species of Candida with the remaining 10% being attributable to Aspergillus, Cryptococcus, and Pneumocystis. While antifungal compounds with some efficacy have been developed, progress in discovery of new antifungal compounds has been slow and lags far behind the rapid growth in the incidence of systemic fungal infections. (See e.g. WO 2004/050685 PCT/US2003/038595 and references therein; Abi-Said et al., 1996; Rees et al., 1998.) This is because of the innate difficulties of finding selective inhibitors for one large group of eukaryotes (fungi) invading another (humans). As a consequence, the current antifungal drug market is dominated by only two classes of drugs, polyenes and azoles, both of which have significant limitations, in terms of efficacy, toxicity, problems with drug-drug interactions and the generation of resistant organisms (e.g. Rex et al., 1995; Hay, 2003). Hence, there is an urgent need for new antifungal compounds with novel modes of action.
Inositolphosphoryl ceramides are sphingolipids found in a number of fungi including but not limited to all major human pathogens (Lester and Dickson, 1993; Vincent and Klig, 1995; Dickson and Lester, 1999). Organisms such as C. albicans, A. fumigatus, C. neoformans and H. capsulatum all contain inositolphosphoryl ceramides, as does S. cerevisiae, S. pombe, and N. crassa. The inositolphosphoryl ceramide biosynthesis pathway in fungi involves at least eight separate reactions, each catalyzed be a specific enzyme. The first five steps in the pathway, starting with the assembly of 3-ketohydrosphingosine, by the enzyme serine palmitoyltransferase, and ending with the hydroxylation of ceramide, by ceramide hydroxylase, are quite similar to the corresponding steps in the mammalian sphingolipid biosynthesis pathway (Nagiec et al., 1997; Dickson and Lester, 1999). Consistent with this, it has been found that inhibitors targeting these reactions have close to equal efficacy towards fungi and mammalian cells and, consequently, such compounds have little potential for development into antifungal drugs. However, the sixth reaction step is unique to fungi and plants. In this step the enzyme IPC synthase catalyzes the transfer of inositol phosphate from phosphatidyl inositol, to ceramide, to form inositol phosporylceramide (FIG. 1). Genetic and mutational studies have demonstrated that this reaction is essential in fungi (Nagiec et al., 1997). It has also been shown, in several organisms, that inhibition of this reaction step is cidal (e.g. Takesako et al., 1993; Endo et al., 1997). By contrast, it has also been demonstrated that the two to three downstream (from IPC synthase) reaction steps, in the fungal sphingolipid biosynthesis pathway, are not essential (Dickson and Lester, 1999).
The uniqueness of the fungal IPC synthase (IPCS) catalyzed reaction, coupled with the fact that the enzyme is essential in fungi, make IPC synthase an attractive target for antifungal drugs. Further supporting this notion is the recent identification of several very potent natural antifungal compounds that all target IPC synthase (e.g., Mandala 1997, Mandala 1998, Zhong 1999, Kurome 2000). Some of these compounds have demonstrated therapeutic activity in animal models, even when delivered orally (Takesako 1993).
In view of the foregoing, inositol phosphorylceramide (IPC) synthase is an important enzyme in fungi and compounds capable of specifically inhibiting this enzyme would have considerable potential to be developed into antifungal drugs and hence meet an immediate, unmet medical need.
A significant impediment to the identification of novel IPC synthase inhibitors with drug candidate potential, is the lack of an assay suitable for robotized screening of large compound libraries (high-throughput screening). Currently used assays are complicated, labor intense and generate poorly reproducible data (e.g. Mandala et al., 1997; Mandala et al., 1998; Ko et al., 1995; Fischl et al., 1999; Aeed et. al., 2004). As such they are completely unsuitable for high-throughput screening efforts.